The object of the invention is nucleotide sequences of bacteria, in particular Gram+ bacteria such as bacteria of the Bacillus type and more particularly nucleotide sequences of the cryIIIA gene for the control of the expression of DNA sequences in a cell host.
The cryIIIA gene codes for a toxin specific for the Coleoptera and is weakly expressed by Bacillus thuringiensis when it is cloned in a low copy number plasmid.
Bacillus thuringiensis is a Gram-positive bacterium which produces significant quantities of proteins in the form of crystals having a toxic activity towards insect larvae. Two groups of crystal proteins are known, based on the amino acid sequences and the toxicity specificities:
1) the class of the Cry toxins (I, II, III, etc . . . ) which have similar structures;
2) the class of the Cyt toxins, which is not related to the Cry class (Hxc3x6fte, H et al. 1989, Microbiol. Rev. 53: 242-255)
These toxins of B. thuringiensis are of general interest for the purpose of the development of bio-pesticides and also in as much as the synthesis of crystal proteins is known to be perfectly co-ordinated with the sporulation phase of the organism, making this organism interesting for the study of genetic regulation in sporulating Gram-positive bacteria.
Various mechanisms implicated in the regulation of the synthesis of the crystal proteins of B. thuringiensis have been described. The high level of expression of these proteins is attributed, at least in part, to the stability of the mRNA. Some authors have attributed the stability of this mRNA to the presence downstream from the gene for the toxin of a structure playing a terminator role which might act as a positive retro-regulator by protecting the 3xe2x80x2 end of the mRNA from degradation by nucleases, thus increasing the half-life of the transcripts (Wong, H. C. et al., 1986 Proc. Natl. Acad. Sci. USA 83: 3233-3237).
A hypothesis has also been put forward concerning the presence of polypeptides implicated in the synthesis of crystal proteins, polypeptides which are supposed to act either by directing the folding of the protein in the form at a protein having a stable conformation or to protect these proteins from proteolytic degradation.
Studies with the electron microscope and biochemical studies of sporulation in B. thuringiensis show that the production of the crystal protein is dependent on sporulation and is located in the mother cell compartment (Ribier, J. et al. 1973 Ann. Inst. Pasteur 124A: 311-344).
Recently, two sigma factors, sigma 35 and sigma 28, which specifically direct the transcription of the cryIA genes have been isolated and characterized. These amino acid sequences exhibit an identity of 88 and 85% with the sigma factors E and K of Bacillus subtilis, respectively (Adams, L. F., 1991, J. Bacteriol. 173: 3846-3854). These sigma factors are produced exclusively in sporulating cells and are capable of functioning in the mother cell compartment, confirming that the expression of the genes for the crystal protein is controlled in time and space. Thus, in the prior art it has been concluded that the expression of the gene with time is, at least in part, ensured by the successive activation of the sigma factors specific for sporulation. Hitherto, three groups of promoters have been identified. Two of these groups include promoters recognized by specific sigma factors and, according to the prior art, the sigma factors associated with the third group of promoters (including that of the cryIIIA gene) have not been identified (Lereclus, D., et al. 1989 American Society for Microbiology, Washington, D.C.).
Finally, the copy number of the plasmid bearing the gene seems to be an important factor for the expression of the cry gene in B. thuringiensis. In the B. thuringiensis wild type strain, the cry genes are localized on large plasmids, present in a low number of copies.
Cloning experiments with a 3 kb HindIII fragment cloned in a low copy number plasmid lead to a low production of toxins in a non-crystal-forming strain (cryxe2x88x92) of B. thuringiensis. On the other hand, large quantities of toxins are synthesized when the gene is cloned in plasmids of high copy number (Arantes, O et al. 1991, Gene 108: 115-119).
The object of the invention is agents making it possible to obtain a high level of expression of the protein encoded in the cryIIIA gene and more generally agents making it possible to control the level of expression of DNA sequences coding for a specific protein of interest in bacterial strains, preferably Gram+ strains such as Bacillus strains, since it is possible to obtain this expression when the coding DNA sequence is located on a vector, in particular on a plasmid of low copy number.
Generally speaking the invention relates to an expression system comprising a DNA sequence, able to intervene in the control of the expression of a coding nucleotide sequence and obtained by associating two distinct nucleotide sequences intervening in different but, preferably, not dissociable ways in the control of the expression of the coding sequence. The first nucleotide sequence exhibits a promoter activity whereas the second sequence, initiated by the promoter activity of the first, intervenes to enhance the expression of the gene. The DNA sequence of the invention makes it possible to attain a high level of expression of the coding part of a gene in a bacterium, in particular a Gram+ type of bacterium.
The first nucleotide sequence of the expression system of the present invention identified in the framework of the present demand as being the promoter consists of either the promoter of the host strain in which the gene of interest to be expressed is introduced, or of an exogenous promoter, functional in the host used. The second nucleotide sequence of the expression system of the invention identified in the present application as being the xe2x80x9cdownstream regionxe2x80x9d designates any sequence preferably situated between the promoter and the sequence coding for a gene to be expressed, able to play a role particularly at the post-transcriptional level when the gene is expressed. More particularly, the downstream region does not act directly on the translation of the coding sequence to be expressed.
In a preferred manner, the xe2x80x9cdownstream regionxe2x80x9d consists of a nucleotide sequence, particularly an S2 sequence or a sequence analogous to S2, containing a region essentially complementary to the 3xe2x80x2 end of the RNA, particularly the 16S RNA, of the ribosomes of bacteria, particularly of Gram+ bacteria of the Bacillus type.
The nucleotides forming the DNA sequence according to the invention may or may not be consecutive in the sequence from which the DNA sequence is defined.
In the context of the present application the expression xe2x80x9cDNA sequence able to intervene in the control of the expression of a coding nucleotide sequencexe2x80x9d expresses the capacity of this DNA sequence to initiate or prevent the expression of the coding sequence or to regulate this expression in particular at the level of the quantity of the product expressed.
A DNA sequence according to the invention is such that the coding nucleotide sequence that it controls is placed immediately downstream, in phase with the same reading frame as it or, on the other hand, it is separated from this DNA sequence by a nucleotide fragment.
Hence the invention relates to a DNA sequence for the control of the expression of a coding sequence for a gene in a cell host, the DNA sequence is characterized in that it includes a promoter and a nucleotide sequence or downstream region situated in particular downstream of the promoter and upstream of said coding sequence. The nucleotide sequence or downstream region contains a region essentially complementary to the 3xe2x80x2 end of a bacterial ribosomal RNA. The DNA sequence of the invention is capable of intervening to enhance the expression of the coding sequence placed downstream in a cell host.
The inventors have identified a DNA sequence of the type previously described, capable of intervening in the control of the expression of the coding sequence of the cryIIIA gene, and making it possible in particular to obtain a high level of expression when the coding sequence is placed on a low copy number plasmid.
The invention also relates to a DNA sequence characterized by the following properties:
it is included in a DNA sequence about 1692 bp long, defined by the restriction sites HindIII-PstI (H2xe2x88x92P1 fragment), such as that obtained by partial digestion of the 6 kb BamHI fragment borne by the cryIIIA gene of Bacillus thuringiensis strain LM79;
it is capable of intervening in the control of the expression of a coding nucleotide sequence placed downstream in a host cell, in particular a bacterial cell host of the Bacillus thuringiensis and/or Bacillus subtilis type.
The restriction sites referred to above are shown in FIG. 1.
In the remainder of the text the abbreviations Hn will be used to designate the HindIII site having the position xe2x80x9cnxe2x80x9d with respect to the first HindIII site of the BamHI fragment. Similarly, the expression Pn designates the PstI site at position xe2x80x9cnxe2x80x9d with respect to the first PstI site an the BamHI fragment.
The DNA sequence defined above can be isolated and purified for example from the plasmid bearing the cryIIIA gene of Bacillus thuringiensis. 
The expression system for cryIIIA comprises a first nucleotide sequence or promoter situated between the TaqI and PacI sites (positions 907 to 990) and a second nucleotide sequence or xe2x80x9cdownstream regionxe2x80x9d included between the XmnI and TaqI sites (positions 1179 to 1559) as shown in FIG. 6. The presence of two sequences of this type is preferred to obtain an optimal level of expression of the cryIIIA gene or of another gene placed under the control of this expression system.
Also included in the framework of the invention is an expression vector characterized in that it is modified at one of its sites by a DNA sequence such as that described above so that said DNA sequence intervenes in the control of the expression of a specific coding nucleotide sequence.
A vector of the invention may preferably be a plasmid, for example a plasmid of the replicative type.
A particularly useful vector is the plasmid pHT7902xe2x80x2lacZ deposited with the CNCM (Collection Nationale de Cultures de Micro-organismesxe2x80x94Parisxe2x80x94France) on Apr. 20th 1993 under No. I-1301.
The object of the invention is also a recombinant cell host characterized in that it is modified by a DNA sequence such as that previously defined or by an expression vector described above. A particularly useful cell host is the strain 407-OA:KmR (pHT305P) deposited with the CNCM on May 3rd 1994 under No. I-1412.
The object of the invention is a DNA sequence capable of influencing the expression of the coding part of a gene in a bacterial cell host. More particularly, the invention relates to the association of two nucleotide sequences, namely a promoter and a downstream region capable of intervening at the post-transcriptional level when the coding part of the gene is expressed.
The expression system of the invention which, as will be described in detail hereafter, probably involves the hybridization of a part of the downstream region with the 3xe2x80x2 end of the 16S RNA of a bacterial ribosome, may be used for the expression of genes in a wide range of host cells. This extensive used of the expression system of the invention is possible, given the considerable homology observed at the level of the various 16S RNAs of bacterial ribosomes. Since the inventors have defined the regions essential for its functioning, the expression system of the present invention can thus be used in any type of bacterial host, the necessary adaptations forming part at the knowledge of the specialist.
In general and without wishing to restrict it for reasons which will become evident below, the expression system of the present invention when used for the expression of genes in Gram+ bacteria of the Bacillus type is situated upstream from the coding part of the gene to be expressed. More particularly, the downstream region is normally situated immediately upstream from the gene whereas the promoter is located upstream from the downstream region, although another position might be envisaged for this latter. It is possible to envisage the displacement of the downstream region when the system is used in a cell host of the E. coli type in which the mRNAs are degraded in the reverse sense. It is also possible to envisage the use of a downstream region downstream and upstream of the coding sequence which would permit the xe2x80x9cprotectionxe2x80x9d of the coding region by a mechanism which will be described in detail below.
According to a first preferred embodiment of the invention, the DNA sequence corresponds to the HindIII-PstI (H2xe2x88x92P1) sequence described above and comprises two nucleotide sequences (a promoter and a downstream region) having distinct functions.
According to a particularly useful embodiment of the invention, the DNA sequence corresponds to the nucleotide sequence designated by the expression SEQ ID NO:1 and corresponding to the DNA fragment comprising the nucleotides 1 to 1692 of the sequence shown in FIG. 3.
The promoter and the downstream region of the DNA sequence of the invention are described in detail below.
Preferably, a DNA sequence of the invention intervenes at the level of the control of transcription.
In this case it is a nucleotide sequence previously identified as being the promoter. Generally speaking as mentioned previously, the promoter is situated upstream from the downstream region and hence at a certain distance from the coding region of the gene. However, it is possible to envisage the relocation of the promoter provided it remains localized upstream from the downstream region.
As to the nature of the promoter, it seems preferable to use a promoter derived from the host cell used for the expression of the gene of interest. However, in certain situations the use of an exogenous promoter may be indicated. For example, promoters such as the promoters of the degO, xcexPL, lacZ, cryI, cryIV or xcex1-amylene genes may be used.
In the context of the present invention particularly preferred fragments comprising a promoter region are the following fragments, shown in FIG. 1:
the sequence defined by the TaqI-PacI restriction sites; for the sake of convenience, PacI is taken to designate the end of this fragment which is in reality found at nucleotide 990 of the sequence shown in FIG. 3, whereas the PacI site ends at position 985,
or any fragment of this sequence, which conserves the properties of this sequence with respect to the control of the expression of coding nucleotide sequence.
More particularly, any part of at least 10 nucleotides of this sequence, naturally consecutive or not, capable of intervening in the control of the expression of a coding nucleotide sequence placed downstream in a cell host constitutes a preferred embodiment of the invention. For example, within the sequence mentioned previously are found the xe2x88x9235 (TTGCAA) and xe2x88x9210 (TAAGCT) boxes of the promoter.
According to another embodiment of the invention the xe2x80x9ccontrolxe2x80x9d DNA sequences comprising the promoter mentioned above are characterized by their nucleotide sequence. In this respect, the object of the invention in particular is the DNA sequences corresponding to the following sequences:
the DNA sequence corresponding to the SEQ ID NO:3 sequence, which corresponds to nucleotides 907 to 990 of the sequence shown in FIG. 3 (SEQ ID NO:1), or a variant comprising the nucleotides 907 to 985.
The object of the invention is also DNA sequences hybridizing under non-stringent conditions, such as those defined below, with one of the sequences described above. In this case, one of the above sequences in question is used as probe.
A sequence of the invention included in the downstream region is selected for its capacity to intervene in order to enhance the expression of a gene which would be initiated by a promoter situated upstream from this sequence. It is probably a sequence capable of intervening at the post-transcriptional level when the coding sequence is expressed.
In fact, the experimental results obtained by the inventors seem to indicate that the post-transcriptional effect of the downstream region previously defined result, at least when the cryIIIA gene is being expressed, from the hybridization between the 16S ribosomal RNA of the host cell and an S2 sequence of the cryIIIA messenger RNA. It seems that the ribosome or a part of the ribosome binds to this downstream region and thus protects the mRNA from exonuclease degradation initiated at the 5xe2x80x2. This binding is thus expected to have the effect of increasing the stability of the messengers and of thus enhancing the level of expression of the cloned gene.
One of the particularly preferred fragments in the context of the embodiment of the invention and one which may be used as downstream region is the following fragment, shown in FIG. 1:
the sequence defined by the restriction sites XmnI-TaqI (positions 1179 to 1556),
or any fragment at this sequence conserving the properties of this sequence with respect to the control of the expression of a coding nucleotide sequence.
According to another embodiment of the invention, the xe2x80x9ccontrolxe2x80x9d DNA sequences comprising the downstream region mentioned above are characterized by their nucleotide sequence. In this respect, the object of the invention is in particular the DNA sequences corresponding to the following sequences:
the DNA sequence corresponding to the sequence SEQ ID NO:4, which corresponds to nucleotides 1179 to 1559 of the sequence shown in FIG. 3 (SEQ ID NO:1),
the DNA sequence corresponding to the sequence SEQ ID NO:5, which corresponds to nucleotides 1179 to 1556 of the sequence shown in FIG. 3 (SEQ ID NO:1),
the DNA sequence corresponding to the sequence SEQ ID NO:11, which corresponds to nucleotides 1413 to 1556 of the sequence shown in FIG. 3 (SEQ ID NO:1),
the DNA sequence corresponding to the sequence SEQ ID NO:8, which corresponds to nucleotides 1413 to 1461 of the sequence shown in FIG. 3 (SEQ ID NO:1),
the DNA sequence corresponding to the sequence SEQ ID NO:9 corresponding to the following DNA fragment:
5xe2x80x2-AGCTTGAAAGGAGGGATGCCTAAAAACGAAGAACTGCA-3xe2x80x2
3xe2x80x2-ACTTTCCTCCCTACGGATTTTTGCTTCTTG-5xe2x80x2
the DNA sequence corresponding to the squence SEQ ID NO:10 corresponding to the following DNA fragment:
5xe2x80x2-CTTGAAAGGAGGGATGCCTAAAAACGAAGAAC-3xe2x80x2
3xe2x80x2-GAACTTTCCTCCCTACGGATTTTTGCTCTTG-5xe2x80x2
The object of the invention is also DNA sequences hybridizing, under non-stringent conditions such as those defined hereafter, with one of the sequences described above. In this case, the relevant sequence defined above is used as probe.
It seems that the downstream region consists initially of a region said to be xe2x80x9cessentialxe2x80x9d, sufficiently complementary to the 3xe2x80x2 end of a 16S bacterial ribosomal RNA to allow the binding of the ribosome to this essential region. Downstream from this essential region bearing the ribosomal binding site, a second region is assumed to be situated comprising an additional structure capable of having an additional positive effect at the level of the expression of the coding sequence. It is possible that this second sequence prevents the movement of the ribosome once this latter is bound to the essential region.
For example, in the expression system of the cryIIIA gene, it seems that the nucleotide sequence situated between the positions 1413 and 1556 of the sequence shown in FIG. 3 comprises the region essential for ribosomal binding as well as the second region downstream from the binding site. Although the second region is not absolutely essential for obtaining an enhanced expression of the coding sequence, it seems that its deletion reduces the expression yields. In fact, experimental results have shown that the deletion of the region situated between the nucleotides 1462 and 1556 of the sequence shown in FIG. 3 leads to a slight diminution of the expression of the coding sequence.
It seems that the minimal length of the nucleotide sequence making possible adequate binding to the ribosome is about 10 nucleotides. The object of the invention is thus also any part of at least 10 nucleotides of the H2xe2x88x92P1 sequence, naturally or not consecutive, capable of controlling in a cell host of the Bacillus type the expression of a coding nucleotide sequence placed downstream or this part of the H2xe2x88x92P1 sequence.
In the specific case of the expression system of the cryIIIA gene, it would seem that the sequence of the xe2x80x9cessentialxe2x80x9d region including the binding site is the following:
5xe2x80x2-GAAAGGAGG-3xe2x80x2
3xe2x80x2-CTTTCCTCC-5xe2x80x2
It is possible to make minor modifications at the binding site in as much as the intensity of the interaction between the 3xe2x80x2 end of the 16S ribosomal RNA and this xe2x80x9cessentialxe2x80x9d region is sufficiently strong for there to be hybridization between the ribosome and the binding site. From the calculations of the interaction energy which may be carried out by the specialist skilled in the art, modifications to the binding site can be envisaged if the intensity of the binding remains about the same as the the intensity measured when the natural xe2x80x9cessentialxe2x80x9d region is used.
In the case of the binding site previously illustrated, it is possible to envisage certain modifications to the first four nucleotides as well as to the seventh nucleotide. However, it seems that the nucleotides in positions 5, 6, 8 and 9 are important for maintaining an appropriate intensity of interaction during hybridization with the 16S ribosomal RNA.
Since the 3xe2x80x2 end of the 16S bacterial in RNA is relatively well conserved from one bacterial species to another, the expression system of the present invention may thus be used in a large number of bacterial hosts without substantial modifications having to be made.
The object of the invention is thus also a DNA sequence characterized by the following properties:
it is contained in a nucleotide sequence hybridizing under non-stringent conditions with the DNA fragment included between the nucleotides 1413 and 1559 of the sequence shown in FIG. 3;
it is capable of intervening in the control of the expression in a host cell of a coding sequence, in particular a sequence coding for a Bacillus polypeptide, toxic towards insects or a sequence coding for a polypeptide expressed during the stationary phase in Bacillus.
A sequence coding for a Bacillus polypeptide, toxic towards insect larvae is for example a sequence included in the cryIIIB gene of B. thuringiensis. 
A DNA sequence corresponding to this definition can be identified by using oligonucleotide primers.
Hybridization under non-stringent conditions between the test DNA sequence and the DNA fragment included between the nucleotides 1413 and 1559 of the sequence of FIG. 3 used as will be conducted as follows:
The DNA probe and the sequences bound to the nitrocellulose filter or to the nylon filter are hybridized at 42xc2x0 C. for 18 h with shaking in the presence of formamide (30%), 5xc3x97SSC of the 1xc3x97Denhardt solution. The 1xc3x97Denhardt solution is composed of 0.02% Ficoll, 0.02% polyyvinylpyrrolidone and 0.02% bovine serum albumin. The 1xc3x97SSC is composed of 0.15M NaCl and 0.015 M sodium citrate. After hybridization, the filter is succesively washed at 42xc2x0 C. for 10 minutes in each of the following solutions:
formamide (30%), 5xc3x97SSC
2xc3x97SSC
1xc3x97SSC
0.5xc3x97SSC
The hybridization conditions just described are those which are used for all the applications of the present invention when necessary.
The DNA sequences according to the invention may be optionally recombinant among themselves or associated on a vector at different sites. In particular, the TaqI-PacI fragment is advantageously associated with the XmnI-TacI fragment with the sequence SEQ ID NO:8. Such sequences have the advantageous property of making possible a high level of expression (up to 60,000 Miller units) of the coding nucleotide sequence, a level of expression which may be observed with the beta-galactosidase gene.
Furthermore, particularly preferred fragments in the context of the embodiment of the invention are the following fragments shown in FIG. 8B:
the sequence defined by the TaqI-TaqI restriction sites,
or any fragment of these sequences conserving the properties of these sequences with respect to the control of the expression of a nucleotide coding sequence.
According to another embodiment of the invention, the DNA sequences referred to above are characterized by their nucleotide sequence. In this respect, the object of the invention is in particular the DNA sequences corresponding to the following sequences:
the sequence SEQ ID NO:2, corresponding to the fragment comprising the nucleotides 907 to 1559 of the sequence shown in FIG. 3 (SEQ ID NO:1),
the DNA sequence corresponding to the sequence SEQ ID NO:6, which corresponds to nucleotides 907 to 1353 (nucleotides 1 to 447 of SEQ ID NO:6) and 1413 to 1556 (nucleotides 448 to 591 of SEQ ID NO:6) of the sequence shown in FIG. 3 (SEQ ID NO:1),
the DNA sequence corresponding to the sequence SEQ ID NO:7, which corresponds to nucleotides 907 to 990 (nucleotides 1 to 84 of SEQ ID NO:7) and 1179 to 1559 (nucleotides 85-465 of SEQ ID NO:7) of the sequence shown in FIG. 3 (SEQ ID NO:1).
The object of the invention is also DNA sequences hybridizing under non-stringent conditions such as those defined above with one of the sequences described above. In this case, one of the above sequences is used as probe.
The DNA sequences of the invention can be isolated and purified from Bacillus, in particular from B. thuringiensis; they can also be prepared by synthesis according to known procedures.
Also included in the framework of the invention are the RNA sequences corresponding to the DNA sequences described above.
The object of the invention is also a recombinant DNA sequence characterized in that it comprises a defined coding sequence under the control of a DNA sequence corresponding to one of the preceding specifications.
The capacity of the DNAs of the invention to intervene in the control of the expression of nucleotide sequences can be verified by implementing the following test:
the DNA sequence of the invention whose capacity to intervene in the control of the expression of a coding sequence it is desired to evaluate is inserted in a low copy number plasmid upstream from a coding nucleotide sequence.
the plasmid thus prepared is used to transform (for example by electroporation) a strain of Bacillus thuringiensis, for example a B. thuringiensis strain HD1 cryxe2x88x92B;
the Bacillus strain thus transformed is cultured under conditions permitting the expression of the coding nucleotide sequence;
the expression product of this coding nucleotide sequence is detected by current qualitative and/or quantitative measuring procedures.
In order to carry out this test, the coding nucleotide sequence should advantageously be the coding sequence of the cryIIIA gene of Bacillus thuringiensis of for example a sequence coding for beta-galactosidase.
Different types a cell host may be used in the framework of the invention. Mention should be made as an example of Bacillus for example Bacillus thuringiensis or Bacillus subtilis. It is also possible to envisage the use of cells such as E. coli. 
In cell hosts capable of sporulating, the coding sequence may be expressed during the vegetative phase or the stationary phase of growth or during sporulation.
A interesting cell host in the framework of the invention may also be constituted by a vegetal or animal cell.
If it is necessary or desired, depending on the nature of the coding nucleotide sequence expressed, a signal sequence can also be inserted in the expression vector of the invention so that the expression product of the coding sequence is exposed at the surface of the cell host, or even exported from this cell host.
In a really interesting manner it will be possible to use strains of Bacillus which have become asporogenic either naturally or as a result of mutation and in particular strains of Bacillus subtilis or Bacillus thuringiensis. 
Since the inventors have demonstrated that the DNA sequences of the invention permit the expression of a defined coding sequence independently of the sporulation phase of strains of the Bacillus type, an asporogenic host may offer the advantage of providing agents of expression of coding sequences to be included in biopesticide compositions whose possible negative effects vis-a-vis the environment would be expected to be attenuated, and even eliminated.
The asporogenic host selected is particularly advantageous for expressing a coding sequence during its stationary phase of growth, when the coding sequence is under the control of one of the sequences of the invention.
In the case of asporogenic strains of Bacillus obtained by mutation, an example illustrating the particular efficacy of this type of strain for the expression of a coding sequence during the stationary phase of growth is the construction of a B. thuringiensis strain mutated in the spoOA gene. A B. thuringiensis strain in which the spoOA gene is inactivated and which bears a gene, for example a gene for an insecticidal toxin cryI, cryII, cryIII or cryIV or also a gene of industrial interest whose expression is placed under the control of the cryIIIA expression system offers advantageous characteristics. In particular, the B. thuringiensis strain 407.OA:KmR (pHT305P) whose construction is described in detail below has at least the following advantages:
a) overproduction of proteins during the stationary phase of growth;
b) the proteins (for example, biopesticides) remain enclosed in the cell and thus would be expected to have an increased persistence in the environment; and
c) the potential problems linked to the dissemination of spores are thus avoided.